Abstract
BackgroundAlthough laccase has a good catalytic oxidation ability, free laccase shows a poor stability. Enzyme immobilization is a common method to improve enzyme stability and endow the enzyme with reusability. Adsorption is the simplest and common method. Modified biochar has attracted great attention due to its excellent performance.ResultsIn this paper, cetyltrimethylammonium bromide (CTAB)-KOH modified biochar (CKMB) was used to immobilize laccase by adsorption method (laccase@CKMB). Based on the results of the single-factor experiments, the optimal loading conditions of laccase@CKMB were studied with the assistance of Design-Expert 12 and response surface methods. The predicted optimal experimental conditions were laccase dosage 1.78 mg/mL, pH 3.1 and 312 K. Under these conditions, the activity recovery of laccase@CKMB was the highest, reaching 61.78%. Then, the CKMB and laccase@CKMB were characterized by TGA, FT-IR, XRD, BET and SEM, and the results showed that laccase could be well immobilized on CKMB, the maximum enzyme loading could reach 57.5 mg/g. Compared to free laccase, the storage and pH stability of laccase@CKMB was improved greatly. The laccase@CKMB retained about 40% of relative activity (4 °C, 30 days) and more than 50% of relative activity at pH 2.0–6.0. In addition, the laccase@CKMB indicated the reusability up to 6 reaction cycles while retaining 45.1% of relative activity. Moreover, the thermal deactivation kinetic studies of laccase@CKMB showed a lower k value (0.00275 min− 1) and higher t1/2 values (252.0 min) than the k value (0.00573 min− 1) and t1/2 values (121.0 min) of free laccase.ConclusionsWe explored scientific and reasonable immobilization conditions of laccase@CKMB, and the laccase@CKMB possessed relatively better stabilities, which gave the immobilization of laccase on this cheap and easily available carrier material the possibility of industrial applications.
Highlights
Laccase has a good catalytic oxidation ability, free laccase shows a poor stability
Characterization analysis The thermogravimetric analysis (TGA) curves are performed to examine the thermal properties of free laccase (FL), CTAB-KOH modified biochar (CKMB) and laccase@CKMB with a constant heating rate of 10 °C/min from 25 to 800 °C under N2 (Fig. 1)
There are two weight-losses from 30 to 150 °C and > 250 °C for the FL, the first weight-loss is corresponding to the removal of structural water and the second weight-loss is corresponding to the pyrolysis of laccase
Summary
Laccase has a good catalytic oxidation ability, free laccase shows a poor stability. The catalytic mechanism of laccase can be roughly described as T1 takes electrons from the oxidized substrate and transfers them to T2/T3; T2/T3 combines with oxygen atom to reduce O2 to H2O [3]. The substrate spectrum of laccase is very rich, including arylamines, aromatic thiols and substituted phenols, which shows the application potential of laccase in the environmental field [4]. Laccase has a good catalytic oxidation ability, free laccase shows extremely high sensitivity to environmental conditions, which means that the stability of laccase is poor under natural conditions. Enzyme immobilization is a common method to improve enzyme stability and endow the enzyme with reusability
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